It is well-known that the properties and characteristics of proteins may be modified by conjugating groups to the protein. For example, U.S. Pat. No. 4,179,337 disclosed proteins conjugated to polyethylene or polypropylene glycols. Generally, such conjugation generally requires some functional group in the protein to react with another functional group in a conjugating group. Amino groups, such as the N-terminal amino group or the ε-amino group in lysine residues have been used in combination with suitable acylating reagents for this purpose. It is often desired or necessary to control the conjugation reaction, such as where the conjugating compounds are attached to the protein and to control how many conjugating groups are attached. This is often referred to as specificity or selectivity.
Site-specific modification of proteins is a longstanding challenge in the pharmaceutical and biotechnology arts. The classic methods oftentimes lead to non-specific labeling (e.g. NHS Lys labeling) or require engineering (e.g. maleimide Cys labeling or unnatural amino acids). In addition, the repertoire of selective chemical reactions, however, is very limited. One alternative is, by recombinant methods, to introduce special unnatural amino acids having a unique reactivity and then exploit this reactivity in the further derivatization. Another alternative is the use of enzymes which recognize structural and functional features of the protein to be modified. An example of this is the use of microbial transglutaminase (mTGase) to selectively modify Gln residues in growth hormone. Other documents disclose the use of transglutaminase to alter the properties of physiologically active proteins. See e.g. EP 950 665, EP 785 276 and Sato, Adv. Drug Delivery Rev., 54, 487-504 (2002), which disclose the direct reaction between proteins comprising at least one Gln and amine-functionalized PEG or similar ligands in the presence of transglutaminase; see also Wada in Biotech. Lett., 23, 1367-1372 (2001), which discloses the direct conjugation of P-lactoglobulin with fatty acids by means of transglutaminase. The reaction catalyzed by the transglutaminase is a transamidation reaction in which the primary amide of the glutamine residue is converted to a secondary amide from a primary amine present in the reaction mixture.
The selective derivatization of proteins remains a very difficult task; the derivatization of lysines in a protein by acylation is an even more inherently non-selective process. Thus, there is at present no efficient method for the selective derivatization of lysine residues. Accordingly, there is a need in the art for methods of selectively derivatizing amino acid residues such as lysine in proteins or polypeptides.